1. Field of the Invention
This invention resides in the field of energy transfer fluorescent labels.
2. Description of the Prior Art
Energy transfer between pairs of covalently linked fluorescent dyes is well known. The advantage of energy transfer is that the combined dyes produce a larger Stokes shift than either of the individual dyes, and this enhances each of the various procedures in which these covalently linked dyes are used as fluorescent labels by producing a clearer and more accurate readout. Included among these procedures are DNA sequencing, gene mapping studies and other nucleic acid fragment analyses, but the technology applies in general to any procedure in which a species of interest, or several distinct species, are labeled for purposes of detection, identification, or separation.
A recent development in the use of energy transfer dye pairs is the use of sets of energy transfer labels were each member of the set has a common donor fluorophore while the acceptor fluorophore differs from one member to the next within the set. Sets of this type are useful in the multiplexing of samples to separate, identify and detect two or more components simultaneously in a single procedure. One procedure in which these sets of labels are particularly useful is the Sanger dideoxy chain-termination method of DNA sequencing, but sets of this type are generally useful in any procedure where fluorescent labels are used to differentiate between components. By virtue of the common donor fluorophore, all labels in the set are irradiated at a single wavelength while fluorescent emissions are detected at distinct wavelengths that permit differentiation between the components. These sets and their uses are disclosed in International Patent Application Publication No. WO 95/21266, published Aug. 10, 1995 (Regents of the University of California, Mathies et al., and Wang, Y., et al., Anal. Chem. 67:1997-1203 (1995).
Selection of the particular donor and acceptor fluorophores to be used in synthesizing the labels and compiling the various members of a set is done with certain considerations. Included among these considerations are the wavelengths of the lasers available for irradiation, and the extinction coefficients, quantum yields, emission spectra, and photostability of the dyes. Unfortunately, donor/acceptor pairs used heretofore are not optimal in all of these properties, and this limits the sensitivity of the labels and their range of use. The best donor/acceptor pairs would be those in which the donor has superior photostability and a high extinction coefficient at the wavelength of available lasers, and in which the rate of energy transfer from the excited donor to the acceptor well exceeds the rates of the other processes that compete for the excited state of the donor, i.e., the natural fluorescence rate of the donor as well as the various radiationless processes that deplete the excited state of the donors.